Predictive value of the uterotrophic assay for genistein carcinogenicity in the neonatal mouse: relevance to infants consuming soy-based formula. (Correspondence).

Markey et al. (1) recently questioned the sensitivity to estrogens of the rodent uterotrophic assay. However, an example of the biological validity of this assay was recently provided by studies on diethylstilbestrol (DES) and genistein by Newbold et al. (2). These investigators used the neonatal mouse uterotrophic assay to define estrogen-equivalent subcutaneous (sc) injection doses of these two chemicals: 1 [micro]g/kg/day DES and 50 mg/kg/day genistein (a difference of 50,000-fold; Table 1). This estrogen-equivalent factor is identical to observations in similarly conducted weanling mouse uterotrophic assays of DES and genistein (Table 1) (3). Newbold et al. (2) also used the same dose levels to evaluate neonatal carcinogenicity of DES and genistein in the mouse uterus; they observed similar incidences of uterine adenocarcinomas with both chemicals in 18-month-old mice (Table 1). This confluence of findings in the neonatal and weanling mouse, and the similar carcinogenic potency of the two chemicals when evaluated at uterotrophic-equivalent dose levels, is encouraging for the utility of the rodent uterotrophic assay for predicting adverse effects in rodents. In particular, the fact that there is insufficient genistein in soy-based infant formula to trigger a uterotrophic response in rodents drinking the formula ad libitum indicates the probable absence of a carcinogenic hazard under normal conditions of use (4). It is of interest that the estrogen-equivalent dose levels of these two chemicals in estrogen receptor binding assays are separated by a factor of only 9,000 (5). The expansion to the 50,000 factor seen in the mouse assays is probably associated with disparities in target tissue dose due to differences in serum protein binding and excretion of the two chemicals in vitro and in vivo, differences that would need to be evaluated separately for humans.

Newbold et al. (2) suggested that the neonatal carcinogenicity of genistein should trigger a close examination of the potential hazard posed to infants by the consumption of soy-based infant formula, because genistein is the major isoflavone phytoestrogen formed from ingestion of soy products. The need for such a review was based on the assumption that 50 mg/kg/day genistein by sc injection, as used in the neonatal carcinogenicity bioassay, was representative of the levels of exposure to genistein experienced by infants fed soy formula (2). Given the potential societal interest in these new data (2), we suggest that the following facts should form a part of any review of the use of soy infant formula. First, genistein shows remarkable differences in route of administration in the uterotrophic assay, with the sc route being substantially more sensitive than the oral route (3). Thus, the use of the sc route by Newbold et al. (2) will inevitably have exaggerated the hazard posed to infants drinking soy formula. Second, the work of Setchell et al. (6), quoted by Newbold et al. (2) to support the relevance to infants of their dose of 50 mg/kg/day genistein, actually estimated that infants drinking soy formula are exposed to 6-11 mg/kg/day total isoflavones, a figure slightly higher than the 4.5 mg/kg/day estimated by the U.K. Food Standards Agency (7). In fact, oral administrations of genistein in this dose range (6,7) are inactive in the uterotrophic assay (3,4,8). Third, the major isoflavone in soy formula is not genistein, but rather its glycone conjugate, genistin (6). This conjugate is transformed into the estrogen genistein in the gut, a transformation attenuated in infants (6). Thus, it is tenuous to relate experimental data derived from the sc injection of genistein to the hazard posed to infants by drinking infant formula containing the glycone genistin. Fourth, the mouse chow used by Newbold et al. (2) (NIH-31) contained 46 [micro]g genistein/g diet, which is similar to the 42 [micro]g genistein/g in the RM1 diet (9) used by Ashby (3). Consequently, the control and test pups used in the carcinogenicity bioassay of genistein were potentially exposed to low levels of genistein in utero via the milk, and in their lifetime by the diet (a mouse weighing 25 g eating 4 g of diet a day would be exposed to ~7 mg/kg/day genistein, together with other isoflavones and their conjugates). The zero incidence of uterine adenocarcinomas in the control animals used by Newbold et al. (2) therefore suggests the existence of a threshold dose for the carcinogenicity of genistein, as observed in the uterotrophic assay (3). A final complication to the proposed safety review on infant formula is that uterotrophic activity and advanced sexual development is observed in rodents exposed to commercial infant formula via their drinking bottles (4). These effects were unrelated to the constituent phytoestrogens but were associated with centrally mediated nutritional influences leading to advanced puberty (4,9).

In conclusion, although the neonatal carcinogenicity of genistein is of significant scientific interest, the test protocol employed in the generation of those data render them of little value for purposes of infant risk assessment. In particular, the exposures to genistein experienced by the mouse neonates may have been several orders of magnitude higher than those experienced by infants drinking soy-based formula.

Table 1. Confluence of the neonatal and weanling mouse uterotrophic
assay data and the neonatal mouse uterine carcinogenicity data
for DES and genistein.

                          DES (1 [micro]g/kg/      Genistein (50 mg/kg/
Period of dosing; assay   day) [~0.002 [micro]g/   day) [~100 [micro]g/
 end point (reference)          neonate]                 neonate]

PND 1-5; uterotrophic     190% uterine weight      202% uterine weight
  assay at PND 5(2)         gain                     gain
PND 20-23; uterotrophic   200% uterine weight      240% uterine weight
  assay at PND 24 (3)       gain                     gain
PND 1-5; incidence of              31%                     35%
  uterine adenoma at
  18 m (2)

All of the data compared involved sc injection of the test agents
at the postnatal days (PND) shown. There were no uterine
adenocarcinomas in the control mice (2).
John Ashby
Jenny Odum
Helen Tinwell
Syngenta Central Toxicology Laboratory
Alderley Park, Cheshire, United Kingdom
E-mail: [e-mail]

REFERENCES AND NOTES

(1.) Markey CM, Michaelson CL, Veson EC, Sonnenschein C Soto AM. The mouse uterotrophic assay: a reevaluation of its validity in assessing the estrogenicity of bisphenol A. Environ Health Perspect 109:55-60 (2001).

(2.) Newbold RR, Banks EP, Bullock B, Jefferson WN. Uterine adenocarcinoma in mice treated neonatally with genistein. Cancer Res. 61:4325-4328 (2001).

(3.) Ashby J. Getting the problem of endocrine disruption into focus: the need for a pause for thought. APMIS 108:805-813 (2000).

(4.) Ashby J, Tinwell H, Odum J, Kimber I, Brooks AN, Pate I, Boyle CC. Diet and the aetiology of temporal advances in human and rodent sexual development. J Appl Toxicol 28:343-347 (2000).

(5.) Fang H, Tong W, Shi LM, Blair R, Perkins R, Branham W, Hass BS, Xie Q, Dial SL, Moland CL, et al. Structure-activity relationships for a large diverse set of natural, synthetic, and environmental estrogens. Chem Res Toxicol 14:280-294 (2001).

(6.) Setchell KDR, Zimmer-Nechemias L, Cai J, Heubi JE. Isoflavone content of infant formulas and the metabolic fate of these phytoestrogens in early life. Am J Clin Nutr 08:1453s-1461s (1998).

(7.) MAFF UK. Plant Oestrogens in Soya-based Infant Formulae. MAFF Food Surveillance Information Sheet 167. London, UK:Ministry of Agriculture, Fisheries and Food, 1998. Available: http://www.foodstandards.gov.uk/ maff/archive/food/infsheet/1998/no167/167phy.htm [cited 16 November 2001].

(8.) Organisation for Economic Co-operation and Development. Unpublished data.

(9.) Odum J, Tinwell H, Jones K, Van Miller JP, Joiner RL, Tobin G, Kawasaki H, Deghenghi R, Ashby J. Effect of rodent diets on the sexual development of the rat. Toxicol Sci 61:115-127 (2001).